Week 21 (03/20/23 - 03/24/23): Discoveries in Optimal Growth Conditions with Deinococcus Species

Introduction: 

 

The goal for this week was to figure out the best ways to grow each bacteria. During the last few weeks, we have had difficulties with multiple species of Deinococcus. Each species would grow in TGY broth, however, the OD600 values were too low for the species to be used in any testing. This leads us to trying different growth methods to see if there are better ways to obtain a higher yield. 

 

Methods:

 

400 ul of TGY was prepared and autoclaved. 200 ul of TGY was prepared, separated into 4 125 ul flasks, and then autoclaved. TGY with agar was heated over a hot plate and poured over plates, which were left out overnight to solidify. Media was

 

Gram staining was done on D. indicus and D. grandis. OD600 values were taken on the nanodrop on D. roseus, D. caeni, D. aquatilis, D. gobiensis, and D. indicus. An inoculation was also performed with D. caeni onto a TGY agar plate. Research was also performed to identify the best form of media to be used with each species, as well as the optimal growth temperature.

 

Antibiotic testing was performed on D. grandis with kanamycin and methicillin, where antibiotic was added to the test tube containing TGY, 50 ul was removed from the tubes, and 50 ul of D. grandis was added to the tubes. The samples were incubated at 30^o C for 48 hours.

 

42 test tubes each containing 5 ml of TGY were autoclaved for 30 minutes. Labels were applied to each tubes in preparation for further antibiotic testing next week.

Results:

Figure 1: Gram staining results on D. indicus (left) and D. grandis (right), viewed on the 100X oil immersion lens.

 

 

Species	OD600 Value	Media	Optimal Growth Temperature
D. roseus	0.15	R2A	28o C
D. caeni	0.08	R2A	28o C
D. aquatilis	0.33	R2A	28o C
D. gobiensis	0.42	Trypticase	28o C
D. indicus	0.07	Trypticase	30o C

Figure 2: OD600 values of the different species of Deinococcus, as well as the ideal media and growth temperatures.

 

 

Antibiotic	Absorbance
Kanamycin	2.12
Methicillin	2.18

Figure 3: Antibiotic testing performed on D. grandis. Concentration of both antibiotics was 50 ug/ml. Absorbance was read on a spectrophotometer at 600nm.

 

Figure 4: D. caeni growth on TGY plate after being incubated at 30^o C for 48 hours.

 

 

Discussion:

 

Gram staining on the species shown in figure 1 did not show any signs of contamination. In regards to the OD600 values taken, all 5 species had a low OD value so we could not perform any antibiotic testing this week. Now that we have identified the best type of media to use for each species, we will inoculate into liquid broth next week and see if we can obtain more growth next week. 

 

Our sample of D. caeni grown on a TGY plate showed signs of contamination, with white colonies that were not expected to grow. This leads us to believe that either the TGY was contaminated, or the original sample of the bacteria was contaminated. We will perform gram staining on the TGY and the original plate of D. caeni to see if we can find the source of contamination so we don’t use the material moving forward.